Massive resources have been expended on the development of potential therapies aimed at reversing the hypovolemia that is common to different manifestations of systemic inflammatory response syndrome (SIRS). Sepsis alone accounts for 750,000 cases per year in the United States, resulting in 200,000 deaths (1). This high mortality results from multi organ dysfunction (MODS), which is associated with organ edema secondary to capillary leak (CL). Patients with significant CL are typically managed by administering resuscitation fluids containing osmolytes (e.g., albumin, starches, or dextrans) in addition to vasopressors and other supportive measures.
Capillary leak, which is present in different conditions such as multiorgan dysfunction (MODS), sepsis, trauma, burn, hemorrhagic shock, post-cardiopulmonary bypass, pancreatitis and systemic capillary syndrome, causes morbidity and mortality among a large number of hospital patients. Capillary leak (CL) is a central component of MODS, secondary to severe sepsis and systemic inflammatory response syndrome (SIRS). It is characterized by increased capillary permeability resulting in interstitial edema and decreased tissue perfusion leading ultimately to organ failure and death. The leak aspect of capillary leak syndrome (CLS) is reflected in both the release of water into the interstitial space and high molecular weight components of serum which ordinarily would be retained within the capillaries.
Hypovolemic states often lead to hypoperfusion of vital organs, causing organ dysfunction and ultimately resulting in morbidity and death (2). Hypovolemia can occur either rapidly, as with hemorrhagic shock, or progressively due to an underlying disease, with both types involving a systemic inflammatory process. In hemorrhagic shock, hypovolemia occurs due to a rapid and sudden loss of intravascular volume. Upon resuscitation, an inflammatory process may be triggered in reperfused tissues (ischemia-reperfusion injury) causing endothelial cell (EC) injury and capillary leak (CL) leading to a secondary hypovolemic state. In sepsis and other diseases, systemic inflammation is triggered by the disease and in a similar sequence leads to EC injury, CL, and ultimately hypovolemic shock.
Resuscitation with plasma volume expanders remains a mainstay in treating hypovolemia, but with mixed results. The efficacy and safety of volume expanders, including both colloids (e.g., albumin and starches) and crystalloids, continue to be topics of intense research and controversy (3,4). The unpredictable effectiveness of albumin as a plasma expander may be linked to the severity of the underlying EC injury (5). Specifically, if the endothelial integrity is compromised such that albumin can readily extravasate, the leaking albumin may exacerbate the oncotic gradient favoring CL, as opposed to reversing it.
Though the biological mechanisms that induce CL syndrome are poorly understood, some evidence indicates the involvement of inflammatory cytokines. Fluid replacement with solutions of human albumin is only marginally effective since it does not stop the loss of albumin into the extravascular space. Albumin is important because it is responsible for plasma oncotic pressure as well as for retaining sodium ions in the blood.
Under normal conditions, albumin contributes to about 80% of the total blood colloid osmotic pressure (6) and is ideally sized such that it extravasates at a low physiologic rate (7). In CL patients, 5% to 20% albumin solutions are often administered to increase circulating blood volume and to augment intravascular osmotic properties. This method of retarding CL makes the tenuous assumption that albumin can maintain its normally low extravasation rate during shock. Clinical data, however, show that the efficacy of albumin is inconsistent at best (8,9). Some have even suggested that resuscitation with albumin may increase mortality in critically ill patients (10).
PEGylation has been used extensively (11,12). Modification of interferon beta-1a with polyethylene glycol prolongs its half-life, resulting in higher antiviral activity (13). There have been studies on the use of PEGylated hemoglobin (PEG-Hb) as a substitute for blood (14,15,16). Large amounts of PEG-Hb, constituting up to 80% vascular volume showed that PEG-Hb is effective in maintaining the hemodynamics and oxygen delivery in the rat (17). These studies suggest that PEG-Hb is safe even at very high doses.
Other colloids have been used to treat capillary leak conditions with varying degrees of efficacy. A variety of heterogeneous (Mr weighted average: 125,000-450,000 Da) starch colloids have been proposed or are in use as substitute for albumin (18). While these compounds are less expensive and more readily available than pooled human albumin, use of starch colloids has been restricted to low doses due to safety issues that severely limit their use. In addition, the high Mr (>1,000,000 Da) moieties within the heterogeneous starch colloids can alter blood rheological properties and cause coagulopathy (19). The relatively homogeneous Pentastarch (Mr=110,000) has been shown to attenuate lung injury in an aortic occlusion reperfusion injury model (20).
In a recent study, MAP and heart rate (HR) did not change favorably when hetastarch (HES) was given in a septic pre-treatment rat model (21). In contrast, favorable changes in MAP (increased) and HR (decreased) were observed in rats pre-treated with polymerized hemoglobin. This occurred despite the fact that, at the same molar concentrations, the colloid osmotic pressure of HES (27 mm/Hg) was higher than the polymerized hemoglobin (21 mm/Hg). Use of the latter as a routine plasma expander is however controversial and is complicated by potential side effects particularly in relation to the kidneys.
Finally, several studies have suggested that albumin has an endothelial anti-apoptotic effect by mediating regulation of cellular glutathione and nuclear Factor Kappa B activation (22,23,24). This may play a significant role in sepsis induced CL particularly in light of a recent report that linked CL in different systemic inflammatory response manifestations to endothelial cell apoptosis (25).
The available albumin today has a molecular weight of 69,000 with a very short half-life (4-6 hours) which can easily leak to the extravascular space in capillary leak conditions such as severe sepsis, pancreatitis, burn and trauma. This leaking can cause worsening edema and/or compartment syndrome. The use of pentastarch and hexastarch are of limited value since they are not for use in pediatric patients and can cause bleeding. Additionally, only 15 cc/kg can be used in patients. Further, the pentastarch and hexastarch have been shown to cause intractable pruritus (itching) after use and the effect lasted for years. In fact, some studies state that the use of albumin as a replacement or as a volume expander is counterproductive since it increases edema by drawing fluid out of the capillaries.
Therefore, there is a great need for a composition and a method to effectively prevent and/or treat hypovolemic conditions which does not have the above-described disadvantages.
In particular, it is to be noted that Hemorrhagic shock (HS) is a leading cause of death following trauma (1a-3a). Early management requires, in addition to controlling the hemorrhage, providing fluid therapy to restore tissue perfusion. The choice of initial fluid therapy can have a significant impact on the outcome. After hemorrhagic shock and resuscitation, nuclear factor-κB (NF-κB) is activated, triggering an inflammatory response, characterized by overproduction of cytokines such as TNF-α, chemokines and cell adhesion molecules which activate endothelial cells (EC), macrophages, neutrophils and other cells (4a). These activated cells (5a, 6a) generate oxidation products such as reactive oxygen species (ROS) which cause vascular damage and capillary leak (CL) (7a-10a). Oxidants and free radicals produced following reperfusion are potent inducers of apoptosis (11a), especially of the EC. Shrinkage of these cells worsens the widening of the inter-endothelial cell gaps and exacerbates the capillary leak (12a) leading to albumin loss. In this environment of oxidative stress with low levels of albumin, endothelial integrity is compromised (32a,34a,35a). Oxidation products, cytokines and vascular depletion, worsened by CL, contribute to vascular unresponsiveness to intrinsic and extrinsic pressors (10a, 13a, 14). These events are summarized in FIG. 11.
In another area of note, recent studies indicate that the type of fluid used in hemorrhagic shock resuscitation affects the physiologic response, the immune response and the systemic inflammatory state.
Crystalloids—Lactated Ringer's (LR) and artificial (synthetic) colloids activate neutrophils and up-regulate cell adhesion molecules; these effects are not seen with albumin or fresh whole blood (10a,11a). Moreover, animals resuscitated with LR or artificial colloids developed significant apoptosis, especially in the lungs and spleen (15a, 16a). Aggressive high volume resuscitation, without controlling the bleeding, can exacerbate the hemorrhage by disrupting the early formed soft thrombi, and by diluting coagulation factors (17a). Conversely, small volume resuscitation using hypertonic saline (7.5%, HTS) alone or in combination with a synthetic colloid is superior to high volume resuscitation, especially in head trauma and in patients at increased risk for developing abdominal or extremity compartment syndrome. However, adverse effects have been reported with small volume HTS used alone or in combination with a synthetic colloid, including hyperchloremic acidosis (18a), and anaphylactoid reactions linked to the colloid component (19a). Other fluids in preclinical testing, such as lactate ethyl pyruvate and ketone based fluids, show less cellular injury and better survival in hemorrhaged animals compared to LR (20a, 21a).
Colloids—The efficacy and safety of colloid plasma expanders, including albumin, are controversial (22a, 23a). Artificial colloids, including starches (24a), have been substituted for albumin in treating capillary leak conditions with varying efficacy. While less expensive and more readily available than human albumin, starch colloids are restricted to low doses because the high Mr (>1,000,000) components alter blood rheological properties and cause coagulopathy (23a). In contrast to albumin, synthetic colloids activate inflammatory and apoptotic processes (25a). Albumin does not increase expression of neutrophil adhesion molecule CD-18, an important step in reperfusion injury, while artificial colloids do (26a). Albumin, which accounts for 80% of blood colloid osmotic pressure (27a), extravasates at a low physiologic rate (28a). In patients with CL, 5% or 25% albumin solutions are administered to increase blood volume and to maintain the oncotic gradient. The efficacy of albumin treatment is variable (29a) and some studies indicate that albumin resuscitation may actually increase mortality (30a). However, a recent randomized double blind controlled clinical study in New Zealand and Australia, involving more than 7000 trauma patients receiving normal saline or 5% albumin, showed no difference in 28 day mortality between the two groups (31a), (study presented by Dr S. Finfer at the 33 rd Congress of Society of Critical Care Medicine, February 2004, Orlando, Fla.).
Albumin as an anti-apoptotic and anti-inflammatory agent—In spite of the conflicting studies of the clinical efficacy of albumin resuscitation, a number of lines of evidence indicate that albumin maintains the integrity of the vascular endothelium (32a-34a) by filling hydrophilic pores of the endothelial surface layer, contributing to their stability (35a). Studies employing human tissue explants in rat skin (36a, 37a) indicate that albumin inhibits endothelial cell apoptosis. Albumin acts as a source of thiol groups (Cys-34); this effect has been demonstrated in septic patients with increases in overall thiol concentration of up to 50% following administration of 200 ml 20% albumin (38a). In vitro mechanistic studies showed that albumin exerts its endothelial anti-apoptotic effect by regulating cellular glutathione and NF-κB deactivation. Physiological concentrations of albumin inhibit TNFα induction by inhibiting NF-κB activation (39a). In a rodent model of HS, 25% albumin resuscitation diminished NF-κB translocation and cytokine-induced neutrophil chemoattractant messenger RNA concentrations (40a).
However, it is also to be noted that albumin is ineffective in hemorrhagic shock. The ineffectiveness of unmodified albumin as a plasma expander in the previous studies (27a, 29a, 30a) may be linked to the severity of the underlying endothelial cell injury. If the endothelial integrity is compromised such that albumin can readily extravasate, the leaking albumin may exacerbate the oncotic gradient favoring capillary leak (41a).